In the process of normal cell proliferation, cell division and its pause occur orderly in accordance with cell cycle, while cancer cell proliferation is characterized by disorder. For this reason, abnormality in the cell cycle control mechanism is supposed to have a direct relation with oncogenesis or malignant alteration of cancer. The cell cycle of a mammalian cell is regulated by a serine/threonine kinase called cyclin-dependent kinase (hereinafter abbreviated as Cdk) family, and Cdk needs to form a complex with the regulatory subunit called cyclin in order to exhibit its enzymatic activity. A cyclin itself also have a family, and each Cdk molecule is considered to regulate the progression of a certain cell cycle by forming a complex with a cyclin molecule which is expressed specifically at the corresponding stage of the cell cycle. For example, D type cyclin, in combination with Cdk4 or Cdk6, regulates the progression of G1 phase, cyclin E-Cdk2 regulates G1/S boundary, cyclin A-Cdk2 regulates the progression of S phase, and cyclin B-cdc2 regulates the progression of G2/M, respectively. In addition, three sub-types D1, D2 and D3 are known as a D type cyclin, and moreover the activity of Cdk is considered to be regulated not only by its association with cyclins but also by phosphorylation/dephosphorylation of Cdk molecules, degradation of cyclin molecules and binding with Cdk inhibitor proteins (Advanced Cancer Research, Vol. 66, pp 181-212(1995); Current Opinion in Cell Biology, Vol. 7, pp 773-780(1995): Nature, Vol. 374, pp 131-134 (1995)).
Cdk inhibitor proteins in a mammalian cell are classified roughly into two kinds due to the differences in structure and property; Cip/Kip family and INK4 family. The former inhibits cyclin-Cdk complex widely, while the latter binds with Cdk4 or Cdk6, thereby specifically inhibiting cyclin-Cdk complex (Nature, vol. 366, pp 704-707(1993); Molecular and Cellular Biology, vol. 15, pp 2627-2681(1995): Genes and Development, vol. 9, pp 1149-1163 (1995)).
For example, P21(Sdi1/Cip1/Waf1) is nominated for a representative example of the former, of which RNA transcription is induced by a cancer repressor gene product, p53.
On the other hand, for example, p16 (INK4a/MTS1/CDK4I/CDKN2) is one of the Cdk inhibitor proteins belonging to the latter. The P16 gene is located on the human chromosome 9p21 which are found to be abnormal with a high frequency in human cancer cells. Actually, many cases of deletion of the p16 gene have been reported in clinical patients. Also, high-frequency of tumorigenesis in a p16 knockout-mouse has been reported (Nature Genetics, vol. 8, pp 27-32(1994); Trends in Genetics, vol. 11, pp 136-140(1995); Cell, vol. 85, pp 27-37(1996)).
Each Cdk regulates the progression of cell cycle by phosphorylation of a certain target protein in a specific phase of cell cycle, and above all, the retinoblastoma (RB) protein is considered to be one of the most important target proteins. The RB protein plays an important role in progression from G1 phase to S phase and is rapidly phosphorylated during the term from late G1 phase to initial S phase. It is considered that this phosphorylation is carried out by cyclin D-Cdk4/Cdk6 complex followed by cyclin E-Cdk2 complex, following progression of cell cycle. The complex composed of hypophosphorylated RB and transcription factor E2F in early G1 phase dissociates when the RB protein becomes hyperphosphorylated. As a result, E2F becomes a transcriptional activator, and at the same time, the suppression of the promoter activity by RB-E2F complex is removed, thus leading to the activation of E2F dependent transcription. At present, the Cdk-RB pathway consisting of E2F, its suppressor RB protein, Cdk4/Cdk6 which repressively regulates the function of RB protein, Cdk inhibitory protein which controls the kinase activity of Cdk4/Cdk6, and D-type cyclin is thought to be an important mechanism to regulate the progression from G1 phase to S phase (Cell, vol. 58, pp 1097-1105(1989); Cell, vol. 65, pp 1053-1061(1991); Oncogene, vol. 7, pp 1067-1074(1992); Current Opinion in Cell Biology, Vol. 8, pp 805-814(1996); Molecular and Cellular Biology, vol. 18, pp 753-761(1998)). In fact, E2F-binding DNA sequence is located, for example, upstream of the sequence of many cell growth-related genes which are important in S phase, and it is reported that in several genes among them the transcription is activated in an E2F-dependent manner during the term from late G1 phase to initial S phase (The EMBO Journal, vol. 9, pp 2179-2184, (1990); Molecular and Cellular Biology, vol. 13, pp 1610-1618 (1993)).
Abnormalities of any factors which relates to in the Cdk-RB pathway such as, for example, deletion of functional pl6, high expression of cyclin D1, high expression of Cdk4 and deletion of functional RB protein are frequently found in human cancer cells (Science, vol. 254, pp 1138-1146(1991); Cancer Research, Vol. 53, pp 5535-5541 (1993); Current Opinion in Cell Biology, Vol. 8, pp 805-814(1996). These are abnormal to such an extent that they tend to promote the progression from G1 phase to S phase, and thus it is obvious that this pathway plays an important role in malignant alteration or abnormal growth of cancer cells.
Previously, as known compounds having an inhibitory activity on Cdk family, a series of chromone derivatives represented, for example, by flavopiridol are known (WO 97/16447, 98/13344); however, the inhibitory activity of those chromone derivatives is not sufficient.